In vitro Selection and in vivo Testing of Riboswitch-inspired Aptamers

Engineered aptamers for new compounds are typically produced by using in vitro selection methods. However, aptamers that are developed in vitro might not function as expected when introduced into complex cellular environments. One approach that addresses this concern is the design of initial RNA pools for selection that contain structural scaffolds from naturally occurring riboswitch aptamers. Here, we provide guidance on design and experimental principles for developing riboswitch-inspired aptamers for new ligands. The in vitro selection protocol (based on Capture-SELEX) is generalizable to diverse RNA scaffold types and amenable to multiplexing of ligand candidates. We discuss strategies to avoid propagation of selfish sequences that can easily dominate the selection. We also detail the identification of aptamer candidates using next-generation sequencing and bioinformatics, and subsequent biochemical validation of aptamer candidates. Finally, we describe functional testing of aptamer candidates in bacterial cell culture. Key features Develop riboswitch-inspired aptamers for new ligands using in vitro selection. Ligand candidates can be multiplexed to conserve time and resources. Test aptamer candidates in bacterial cells by grafting the aptamer back onto its expression platform.

This protocol is used in: Nucleic Acids Res. (2023), DOI: 10.1093/nar/gkac1218 Engineered aptamers for new compounds are typically produced by using in vitro selection methods. However, aptamers that are developed in vitro might not function as expected when introduced into complex cellular environments. One approach that addresses this concern is the design of initial RNA pools for selection that contain structural scaffolds from naturally occurring riboswitch aptamers. Here, we provide guidance on design and experimental principles for developing riboswitch-inspired aptamers for new ligands. The in vitro selection protocol (based on Capture-SELEX) is generalizable to diverse RNA scaffold types and amenable to multiplexing of ligand candidates. We discuss strategies to avoid propagation of selfish sequences that can easily dominate the selection. We also detail the identification of aptamer candidates using next-generation sequencing and bioinformatics, and subsequent biochemical validation of aptamer candidates. Finally, we describe functional testing of aptamer candidates in bacterial cell culture.

Graphical overview Background
Aptamers are ligand-binding oligonucleotides that are becoming increasingly useful for broad applications in diagnostics, therapeutics, and synthetic biology (Keefe et al., 2010;Topp and Gallivan, 2010;Wang et al., 2019). Aptamers occur naturally in the context of riboswitches, where they monitor the concentration of a target ligand and manipulate the folding of an adjoining expression platform to control the expression of their associated genes (Sherwood and Henkin, 2016; Kavita and Breaker, 2023). Engineered aptamers that bind different ligands can be developed using a technique called in vitro selection (Ellington and Szostak, 1990; Tuerk and Gold, 1990). This process involves generating a large combinatorial pool of oligonucleotides, selecting for those that bind a target ligand, and amplifying the selected oligonucleotides. This process is repeated iteratively until aptamers for the target ligand are identified. Ideally, engineered aptamers could be useful for intracellular applications. However, the physiochemical conditions inside of a cell differ substantially from that of a test tube. Thus, aptamers developed in vitro might fail to perform inside of a cell, likely due to intrinsic factors such as the failure to reliably fold into the structure required to form the ligand binding pocket (Filonov et al., 2014). To address this, researchers have exploited the architectures of natural riboswitch aptamers to provide scaffolds for combinatorial RNA pools (Porter et al., 2017;Dey et al., 2022;Mohsen et al., 2023). We recently reported the Graftamer approach (Mohsen et al., 2023), in which engineered aptamers that contain a natural riboswitch scaffold are grafted back onto the natural expression platform (Figure 1). Using this approach, we developed aptamers for quinine and caffeine that retain the Guanine-I riboswitch (Mandal et al., 2003) scaffold from the initial combinatorial pool. These aptamers were each grafted back onto the expression platform of a Bacillus subtilis xpt-pbuX Guanine-I riboswitch. The resulting engineered quinine and caffeine riboswitches each display ligand-mediated gene regulation in B. subtilis cultures, indicating that the quinine and caffeine aptamers are functional in cells.

Figure 1. Overview of the Graftamer approach.
First, a combinatorial RNA pool is designed by inserting regions of random RNA sequence (Nx, Ny, and Nz) in between structural features of a natural riboswitch aptamer. The depicted riboswitch contains an aptamer with paired elements P1, P2, and P3. Second, in vitro selection is performed to develop riboswitch-inspired aptamers that bind ligands different than that of the natural riboswitch. Third, validated aptamers that maintain the structural features of the natural riboswitch are grafted back onto the expression platform of the natural riboswitch to construct engineered riboswitches. Fourth, plasmids containing engineered riboswitches positioned upstream of a reporter gene are transformed into a suitable model organism. In the depicted example, the riboswitch functions as an OFF switch. Increased ligand concentration reduces the expression of a lacZ reporter gene, resulting in a corresponding decrease in blue color in the presence of X-gal indicator.
Here, we provide a protocol for this approach, beginning from the design and generation of the initial RNA pool. The selection process described herein is based on Capture-SELEX (Nutiu and Li, 2005;Stoltenburg et al., 2012;Yang et al., 2016;Lauridsen et al., 2018;Boussebayle et al., 2019), wherein the RNA pool is hybridized to a 3′biotinylated DNA capture oligonucleotide, which itself is immobilized on a streptavidin-agarose column ( Figure 2). In principle, RNA molecules that undergo a structural change upon binding a ligand are able to release from the capture oligonucleotide (Nutiu and Li, 2005). However, selfish molecules that slowly release from the capture oligonucleotide in a ligand-independent manner can jeopardize the selection (Mohsen et al., 2023). To counter the proliferation of selfish molecules, we recommend stringent washing and relatively short incubation times. Contamination between parallel lines of in vitro selection poses a threat as well. Thus, we suggest designing a different set of primers for each selection line performed in the same laboratory space. We anticipate that this protocol might have reduced utility for researchers pursuing aptamers of compounds that occur naturally in the target organism or that are unable to accumulate to appreciable intracellular concentrations (e.g., due to rapid efflux or metabolism, or an inability to permeate the cell wall or membrane). Nevertheless, we expect that this protocol will be broadly useful for improving best practices for in vitro selection and for increasing the likelihood of success for other researchers working to develop novel aptamers.

TBE (10× concentrated, 4 L)
To a 4 L flask, add 432 g of Tris base, 220 g of boric acid, 14.9 g of EDTA, and deionized H2O to 4 L. Stir to mix. Filter particulates. Sterilize by autoclaving. The final solution is a 10× concentration buffer containing 0.9 M Tris, 0.9 M borate, and 10 mM EDTA pH 8.0 at ~20 °C.

TAE (50× concentrated, 1 L)
To a 1 L flask, add 242 g of Tris base, deionized H2O to ~800 mL, 57.1 mL of glacial acetic acid, and 100 mL of 0.5 M EDTA pH 8.0 at ~20 °C. Add deionized H2O to 1 L. Filter particulates. Sterilize by autoclaving. The final solution is a 50× concentrated buffer containing 2 M Tris, 1 M acetate, and 50 mM EDTA pH 8.0 at ~20 °C.     a. Based on crystallographic data, determine which nucleotides interact with the natural ligand. b. Randomize nucleotides that interact with the natural ligand, as well as nucleotides that are not required to form conserved tertiary structural interactions. A greater number of randomized nucleotides provides a larger sequence space, though we have performed successful selections with as few as 23 randomized nucleotides. 3. Design a 3′-biotinylated DNA capture oligonucleotide.
a. The capture oligo should be 12-18 nucleotides in length and should contain 10-15 nucleotides of complementarity with a constant region in the RNA pool. b. The capture oligo can be designed to compete with the P1 stem of the RNA pool. c. The capture oligo should contain a 3′-biotin or 3′-biotin-triethylene glycol (TEG) modification. The TEG spacer provides additional space between the RNA/capture oligo hybrid and the streptavidin/biotin complex. We have performed successful selections using capture oligos with and without the TEG spacer. 4. Design primer-binding regions and primers for polymerase chain reaction (PCR).
a. Design forward and reverse primer-binding regions into the RNA pool that have roughly the same melting temperature (Tm). Avoid natural sequences from common laboratory model organisms because this can lead to amplification of nucleic acid contaminants. b. Design a forward primer (sense sequence) that starts with a T7 RNA polymerase (RNAP) promoter sequence at the 5′ end. The T7 RNAP promoter sequence with two additional G nucleotides at the 3′ terminus (for increased transcription efficiency) is as follows: TAATACGACTCACTATAGG. RNA transcribed from this template will start with two G nucleotides. Add the sense DNA sequence corresponding to the forward primer-binding region from the RNA pool after these two G nucleotides. c. Design a primer (reverse primer) that is the reverse complement DNA sequence of the 3′ primerbinding region from the RNA pool. 5. Design an oligodeoxynucleotide template pool.
a. Design a single-stranded reverse complement DNA that contains ~15 base pairs of overlap with the forward primer (not including the T7 RNAP promoter sequence) ( Figure 2, Generate Pool Generation). This strand contains randomized nucleotides (N). Hand-mixed phosphoramidites provide an even distribution between all four nucleotides, but machine mixing is typically more cost effective and is sufficient for this selection protocol. 6. Order and purify custom oligodeoxynucleotides.
a. Longer strands such as the template pool should be ordered at a synthesis scale (≥ 200 nmol) that ensures a sufficient quantity of full-length material. Shorter strands such as forward primer, reverse primer, and capture oligonucleotide can be ordered at the smallest synthesis scale. b. We typically order custom oligodeoxynucleotides with standard desalting and purify in house by denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE). Alternatively, oligodeoxynucleotides could be ordered with PAGE purification. 7. Synthesize the generation zero (G0) double-stranded DNA template by primer extension.  o. Dry the pellet by centrifugal evaporation (speed-vac) on medium heat for 5 min or until dry. Alternatively, the pellet can be air dried. p. Resuspend the pellet in 50 μL of dH2O. Quantitate the concentration of the resulting solution using a NanoDrop spectrophotometer.

B. In vitro selection
1. Hybridize RNA pool to capture oligonucleotide. a. Prepare the following mixture in a 0.5 mL or 1.5 mL tube: Component Quantity 10× selection buffer 10 μL RNA pool x μL* Capture oligonucleotide (10 μM) y μL** dH2O 90 -xy μL *For round one, input 100-1,000 pmol RNA. For subsequent rounds, add 1-10 pmol RNA. **Add a 10× molar excess of capture oligonucleotide relative to RNA pool input. b. Incubate the tube at 90 °C for 1 min and then allow to cool at room temperature for at least 5 min.

Selection:
a. While the RNA-capture oligonucleotide solution is cooling, prepare a column for selection. Add 100 μL of streptavidin-agarose to a Micro Bio-Spin column using a P1000 pipette (the wider tip openings transfer the bead solution more accurately). Place the column in a 1.5 mL tube. b. Prepare an air pressure control device ( Figure 3A). Attach a 27 G × 1/2 needle to a 3 mL syringe.
Then, poke the needle through the center of a Micro Bio-Spin cap. Pull the syringe plunger until it is fully extended. c. Holding the column in one hand and the air pressure control device in the other, place the cap of the device on top of the column with sufficient pressure to create a seal, without shutting the tube. Apply pressure by pressing down on the syringe plunger. This should drain the storage buffer into the collection tube, while the streptavidin-agarose beads remain in the column. d. Wash the column with 100 μL of 1× selection buffer six times to equilibrate the column in selection buffer ( Figure 3B). Each wash is executed by gently pipetting 100 μL of buffer onto the column resin and subsequently using the air pressure control device to drain the buffer. Two consecutive washes can be collected in a single 1.5 mL tube, after which the column should be transferred to a new collection tube. After six washes, transfer the column to a new 1.5 mL collection tube.

Published: Jul 05, 2023
e. Briefly centrifuge the RNA-capture oligonucleotide solution (after cooling for at least 5 min) and apply the entire 100 μL solution to the column. Use the air pressure control device to push the solution through the resin to the collection tube. To maximize the quantity of biotinylated capture oligonucleotide bound to the streptavidin column, re-apply the eluate to the column two additional times. f. Wash the column with 100 μL of 1× selection buffer 10 times to remove RNA molecules retained by nonspecific interactions. In this case, each wash is performed by gently pipetting 100 μL of 1× selection buffer on top of the column resin and then using the air pressure control device to push the solution through, such that it saturates the resin but does not go through into the collection tube. After incubating for 30 s, use the air pressure control device to drain the solution into the collection tube. g. Incubate three times with a solution of the chosen ligand candidate(s) in 1× selection buffer for 30 s.
It is critical that the incubations with ligand solution are performed identically to the washes described in the preceding step, with the only difference being the presence of the ligand candidates. h. Combine the ligand pool eluates and transfer to a Vivaspin 10 kDa molecular weight cutoff column.
The mass of RNA pool molecules is expected to be > 10 kDa and should be retained by the column. i. Centrifuge at 12,000× g for 15 min at room temperature (~20 °C). j. Discard flowthrough. Add 300 μL of dH2O to the column and centrifuge again at 12,000× g for 15 min at room temperature. k. Recover the concentrated RNA (typically < 15 μL) by aspirating with a gel-loading tip.

Synthesize complementary DNA (cDNA) by reverse transcription.
a. During the first round of selection, prepare a generation zero (G0) marker. In parallel with the steps described below, perform reverse transcription using 2 pmol of the initial RNA pool. b. To a 0.5 mL tube, add up to 12 μL of the concentrated RNA, 1 μL of 10 mM dNTPs, and 1 μL of 2 μM reverse primer (2 pmol vii. Hold at 10 °C c. Take a 5 μL aliquot from the completed PCR reaction and mix it with 1 μL of 6× purple loading dye in a new tube. d. To prepare the G0 marker, add 10 μL of 6× purple loading dye to the PCR tube and optionally transfer to a 1.5 mL tube for easier storage at 4 °C or -20 °C. e. Prepare a 1.5% agarose gel by dissolving 0.6 g of agarose in 40 mL of TAE in a 250 mL Erlenmeyer flask. Microwave the solution in bursts with frequent stirring until the agarose is completely dissolved. Just before casting the gel, add 2 μL of ethidium bromide and mix by swirling. f. Load 5 μL each of 100 base pair ladder, G0 marker, and the PCR solution from the current generation in consecutive lanes. g. Run the gel at 115 V for 20 min. Visualize the resulting gel using a Bio-Rad Gel Doc Go Gel imaging system or an equivalent instrument. The bands resulting from the G0 marker and from the current generation should appear to have migrated the same distance, indicating that the amplicons originated from the pool and not from a contaminant. h. Purify the PCR product using a QIAquick PCR purification kit or equivalent. i. Quantitate the concentration of DNA using a NanoDrop spectrophotometer. Convert the concentration from ng/μL to μM by dividing by the approximate molecular weight of the DNA construct.  a. Perform iterative rounds of selection until the pool is sufficiently enriched. After the first few rounds, the researcher can optionally decide to decrease the concentration of ligand candidates to apply additional selection pressure. Another option that can be applied concurrently is to decrease the quantity of input RNA in later selection rounds. b. Typically, the RNA pool will be sufficiently enriched after 8-12 rounds of selection. Enrichment can be assessed by an elution profile, which is performed similarly to the selection process described above.

C. Elution profile
Note: This step involves handling radioactive materials ( 32 P). If the researcher's laboratory is not equipped to handle radioactive materials, the entire process could be performed with unlabeled RNA. In this case, qRT-PCR could alternatively be used to determine relative quantities of RNA eluted with each ligand candidate. 1. Prepare the following mixture in a 0.5 or 1.5 mL tube: Component Quantity 10× selection buffer 10 μL 5′ 32 P-labeled RNA pool x μL (~50,000 counts per minute) Capture oligonucleotide (10 μM) 1 μL dH2O 89 -x μL 2. Incubate the tube at 90 °C for 1 min and then allow to cool at room temperature for at least 5 min. 3. While the RNA-capture oligonucleotide solution is cooling, prepare a column for the elution profile. Add 100 μL streptavidin-agarose to a Micro Bio-Spin column using a P1000 pipette. Place the column in a 1.5 mL tube. 4. Prepare an air pressure control device: attach a needle to a 3 mL syringe. Then, poke the needle through the center of a Micro Bio-Spin cap. Pull the syringe plunger until it is fully extended. 5. Holding the column in one hand and the air pressure control device in the other, place the cap of the device on top of the column with sufficient pressure to create a seal, without shutting the tube. Apply pressure by pressing down on the syringe plunger. This should drain the storage buffer into the collection tube, while the streptavidin-agarose beads remain in the column. 6. Wash the column with 100 μL of 1× selection buffer six times to equilibrate the column in selection buffer.
Each wash is executed by gently pipetting 100 μL of buffer onto the column resin and subsequently using the air pressure control device to drain the buffer. Two consecutive washes can be collected in a single 1.5 mL tube, after which the column should be transferred to a new collection tube. After six washes, transfer the column to a new 1.5 mL collection tube. 7. Briefly centrifuge the RNA-capture oligonucleotide solution (after cooling for at least 5 min) and apply the entire 100 μL solution to the column. Use the air pressure control device to push the solution through the resin to the collection tube. To maximize the quantity of biotinylated capture oligonucleotide bound to the streptavidin column, re-apply the eluate to the column two additional times. After applying and eluting the solution three times, label the tube containing the final eluate as Unbound RNA. 8. Incubate the column with 100 μL of 1× selection buffer for 30 s to remove RNA molecules retained by nonspecific interactions. Each incubation is performed by gently pipetting 100 μL of 1× selection buffer on top of the column resin and then using the air pressure control device to push the solution through, such that it saturates the resin but does not go through into the collection tube. After incubating for 30 s, use the air pressure control device to force the solution through into the collection chamber. Repeat this step until eluates reach background radiation, as assessed by a Geiger counter (typically 6-8 incubations). Collect each eluate in a separate tube. 9. Incubate the column with a 100 μL solution of one of the chosen ligand candidates in 1× selection buffer.
It is critical that the incubations are performed identically to the negative selection steps above, with the only difference being the presence of the ligand candidate. Repeat this step two times, collecting each eluate in a separate tube. If you performed selection with only one ligand candidate, the elution profile, skip to step C12.

E. Biochemical validation of aptamer candidates
1. Perform an elution profile as described in Section C using a single aptamer candidate instead of an RNA pool. For a valid aptamer candidate, we typically expect to observe an increase in signal for only one of the compounds. 2. There are various biochemical methods that can be used to further validate aptamer-ligand binding, including in-line probing (Soukup and Breaker, 1999), selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) (Merino et al., 2005), isothermal titration calorimetry (Slavkovic and Johnson, 2023), and surface plasmon resonance (Arney and Weeks, 2022). Our lab prefers in-line probing, for which 5′ 32 P-labeled RNAs already prepared for the elution profile can be utilized. Refer to previously reported protocols for in-line probing (Regulski and Breaker, 2008). Techniques such as in-line probing and SHAPE that provide structural information are especially useful because they can confirm whether the engineered aptamers retain the structural features of the riboswitch aptamer from which they were derived. With the user's preferred method for biochemical validation, test candidate aptamers for binding with the target ligand and confirm that disruptive mutant(s) display reduced binding.
F. Grafting aptamer candidates onto their expression platforms and functional testing in cells 1. Starting from the sequence of the natural riboswitch that was chosen in part A, remove the entire aptamer domain as well as 2-3 base pairs in the P1 stem located adjacent to the aptamer domain ( Figure 1). 2. Remove the primer-binding regions from the sequence of a biochemically validated aptamer, as well as the sequence of the P1 stem, except for the 2-3 base pairs immediately adjacent to the aptameric core. The number of base pairs retained from the aptamer sequence should be equal to the number that is removed from the expression platform sequence. 3. Graft the engineered aptamer sequence onto the natural expression platform. 4. Place a common promoter sequence (e.g., thiC for E. coli, lysC for B. subtilis) upstream of this engineered riboswitch. 5. Using molecular cloning techniques, install this sequence upstream of a lacZ reporter gene within an appropriate plasmid. Another reporter gene, such as green fluorescent protein or luciferase can optionally be used. 6. Transform this plasmid into a model organism that naturally contains the original riboswitch. 7. Test the function of the engineered riboswitch by culturing the transformed cells in media supplemented with X-gal (100 μg/mL). If the target ligand does not occur naturally in the cell (e.g., a drug compound), supplement different cultures with and without ligand. Differential blue color between cultures with or without the target ligand indicates that the riboswitch is functional. 8. Confirm that the directionality of the chosen riboswitch (ON or OFF switch) is reflected in the observed result. Additionally, confirm that the switching effect diminishes between cultures that contain a disruptive mutant construct.

Data analysis
Analysis of next-generation sequencing data as described in this protocol requires some familiarity with bash commands. Other software, such as FASTAptameR 2.0 (Kramer et al., 2022), can be accessed via the web to facilitate this analysis.

Validation of protocol
To validate the function of engineered quinine and caffeine riboswitches, we quantitated specific β-galactosidase activity using a Miller assay in the presence and absence of the ligand (Mohsen et al., 2023). Three technical replicates were performed. Statistical analysis was performed with a t-test (two-tailed distribution, two sample equal variance).